Recent advances in various scientific and industrial fields have dramatically increased the desirability of synthesizing new chemical and biological substances, and have similarly increased the need to analyze these substances, e.g., to identify their active components, verify their stability, and optimize processes for their manufacture. In an effort to accelerate these capabilities, researchers have sought to introduce a higher degree of automation to synthetic and analytical processes as well as increase the number of processes performed in parallel. Most of these processes are performed with fluids (e.g., liquids and/or gases).
To increase the efficiency of fluid processing performed in parallel, it would be desirable to reduce the number of expensive fluid supply components, such as pumps, valves, regulators, and pulse dampers. Providing common fluid supply components and fluidic splitting networks for supplying common fluid(s) to multiple process regions would appear to address such efficiency concerns. If common fluid supply components are used for parallel fluid processing systems, however, another concern is ensuring that each process region is subject to reproducible process conditions. For example, if it is desired to evenly split common supplies of solvents or reagents to multiple process regions, it may be difficult to ensure that each process region receives a substantially equal flow. The problem may be exacerbated by the presence of solid materials, such as catalysts or separation media, e.g., due to variations in solid particle types, sizes, and/or packing density. Additional concerns may arise if it is desired to vary the composition of common solvents or reagents over time, since it can be difficult to ensure that each process region is subject to the same supply fluid composition at substantially the same time. While instrumentation and flow control devices might be added to each process region to address these problems, adding such components can rapidly increase the complexity and cost of parallel fluid processing systems to the point that they are no longer economical to build, operate, or maintain. Moreover, if microfluidic process regions are used, it can be difficult to accurately measure extremely low fluid flow rates supplied to individual process regions.
In the absence of systems or methods for ensuring that reproducible process conditions are maintained in parallel fluid processing systems, it can be difficult to draw confident conclusions from data obtained from individual process regions in different experimental runs, let alone compare data obtained from multiple process regions in the same experimental run. As a result, the scientific utility of efficient parallel fluid processing systems appears to be limited.
The following example is provided to illustrate one type of fluid processing system and issues associated therewith.
One useful analytical process called “chromatography” is routinely performed in various industrial and academic settings. Chromatography encompasses a number of methods for separating various components of mixtures. Liquid chromatography (“LC”) is a physical method of separation wherein a liquid “mobile phase” (typically consisting of one or more solvents) carries a sample containing multiple constituents or species through a separation medium or “stationary phase.” Various types of mobile phases and stationary phases may be used. Stationary phase material typically includes a solid material disposed within a tube or other boundary. To provide increased surface area so as to enhance separation efficiency, the solid material may be in the form of packed granules (particulate material). The packed material contained by the tube or similar boundary is commonly referred to as a “separation column.” High pressure is often used to obtain a close-packed column with minimal voids between adjacent particles, since better resolution during use is typically obtained from more tightly packed columns. As an alternative to packed particulate material, a porous monolith or similar matrix may be used. So-called “high performance liquid chromatography” (“HPLC”) refers to efficient separation methods that are typically performed at high operating pressures.
Typical interactions between stationary phases and solutes include adsorption, ion-exchange, partitioning, and size exclusion. Commonly employed stationary phase base materials include silica, alumina, zirconium, or polymeric materials. A stationary phase material may act as a sieve to perform simple size exclusion chromatography, or the stationary phase may include functional groups (e.g., chemical groups) to perform other (e.g., adsorption or ion exchange separation) techniques.
Mobile phase is forced through the stationary phase using means such as one or more pumps, voltage-driven electrokinetic flow, gravitational force, or other established means for generating a pressure differential. After sample is injected into the mobile phase, such as with a conventional loop valve, components of the sample will migrate according to interactions with the stationary phase and the flow of such components are retarded to varying degrees. Individual sample components may reside for some time in the stationary phase (where their velocity is essentially zero) until conditions (e.g., a change in concentration of a mobile phase solvent) permit a component to emerge from the column with the mobile phase. In other words, as the sample travels through voids or pores in the stationary phase, the sample may be Separated into its constituent species due to the attraction of the species to the stationary phase. This attraction may be overcome due to, for example, a change in mobile phase composition. The time a particular constituent spends in the stationary phase relative to the fraction of time it spends in the mobile phase will determine its velocity through the column. Following separation in an LC column, the eluate stream contains series of regions having an elevated concentration of individual component species, which can be detected by various detection techniques to identify and/or quantify the species
As illustrated in FIG. 1, a separation column for use in a conventional hydrostatic pressure-driven chromatography system is typically fabricated by packing particulate material 14 into a tubular column body 12. A conventional column body 12 has a high precision internal bore 13 and is usually manufactured with stainless steel, although materials such as glass, fused silica, and/or PEEK are also used. Various methods for packing a column body may be employed. In one example, a simple packing method involves dry-packing an empty tube by shaking particles downward with the aid of vibration from a sonicator bath or an engraving tool. A cut-back pipette tip may be used as a particulate reservoir at the top (second end), and the tube to be packed is plugged with parafilm or a tube cap at the bottom (first end). Following dry packing, the plug is removed and the tube 10 is then secured at the first end with a ferrule 16A, a fine porous stainless steel fritted filter disc (or “frit”) 18, a male end fitting 20A, and a female nut 22A that engages the end fitting 20A. Corresponding connectors (namely, a ferrule 16B, a male end fitting 20B, and a female nut 22B) except for the frit 18 are engaged to the second end to secure the dry-packed tube 12. The contents 14 of the tube 12 may be further compressed by flowing pressurized solvent through the packing material 14 from the second end toward the first (frit-containing) end. When compacting of the particle bed has ceased and the fluid pressure has stabilized, there typically remains some portion of the tube 13 that does not contain densely packed particulate material. To eliminate the presence of a void in the column 10, the tube 13 is typically cut down to the bed surface (or a shorter desired length) to ensure that the resulting length of the entire tube 12 contains packed particulate 14, and the unpacked tube section is discarded. Thereafter, the column 10 is reassembled (i.e., with the ferrule 16B, male end fitting 20B, and female nut 22B affixed to the second end) before use.
A conventional pressure-driven liquid chromatography system utilizing a column 10 is illustrated in FIG. 2. The system 30 includes a solvent reservoir 32, at least one (preferably two) high pressure pump(s) 34, a pulse damper 36, a sample injection valve 38, and a sample source 40 all located upstream of the column 10, and further includes a detector 42 and a waste reservoir 44 located downstream of the column 10. The high pressure pump(s) 34 pressurize mobile phase solvent from the reservoir 32. A pulse damper 36 serves to reduce pressure pulses caused by the pump(s) 34. The sample injection valve 38 is typically a rotary valve having an internal sample loop for injecting a predetermined volume of sample from the sample source 40 into the solvent stream. Downstream of the sample injection valve 38, the column 10 contains stationary phase material that aids in separating species of the sample. Downstream of the column 10 is a detector 42 for detecting the separated species, and a waste reservoir 44 for ultimately collecting the mobile phase and sample products. A backpressure regulator (not shown) may be disposed between the column 10 and the detector 42. Many components of the system 30 are precision manufactured, thus elevating the cost of a typical high performance liquid chromatography system 30 to approximately $20,000–$30,000 or more. Owing to the ever-increasing demand for chromatographic separations, once such a system 30 is purchased, it is often operated on a nearly continuous basis.
The system 30 generally permits one sample to be separated at a time in the column 10. Due to the cost of conventional tubular chromatography columns, they are often re-used for many (e.g., typically about one hundred or more) separations. Following one separation, the column 10 may be flushed with a pressurized solvent stream in an attempt to remove any sample components still contained in the stationary phase material 14. However, this time-consuming flushing or cleaning step does not always yield a completely clean column 10. This means that, after the first separation performed on a particular column, every subsequent separation may potentially include false results due to contaminants left behind on the column from a previous run. Eventually, columns become fouled to the point that they are no longer useful, at which point they are removed from service. A spent column is removed from the system 30 by disengaging threaded fittings, and a new column 30 must be carefully connected via the same threaded fittings to prevent unintended leakage.
To provide increased throughput without concomitantly increased cost, a conventional chromatography system may be modified to split the flow from a common set of one or more (typically two) high pressure pumps to several chromatography columns. Such a system 50 is illustrated in FIG. 3. Mobile phase solvent from a solvent reservoir 52 is pressurized by one or more common high pressure pumps 54, and pressure variations caused by the common pump(s) 54 are damped by a common pulse damper 56. Downstream of the pulse damper 56, the solvent flow is split among multiple columns 10A, 10B, 10N each having an injection valve 58A, 58B, 58N and sample source 60A, 60B, 60N. (Although FIG. 3 shows three columns 10A, 10B, 10N, it will be readily apparent that any number of columns 10A, 10B, 10N may be provided. For this reason, the designation “N” is used to represent the last column 10N, with the understanding that “N” represents a variable and could represent any desired number of columns.) Downstream of each column 10A, 10B, 10N is a detector 62A, 62B, 62N and a waste reservoir 64A, 64B, 64N.
Compared to the system 30 described in connection with FIG. 2, the enhanced system 50 permits significantly higher throughput, since several samples can be analyzed in parallel. Additionally, in the system 50, this increased throughput comes at a lower cost per column/analysis, since the cost of expensive solvent delivery components (particularly the high pressure pumps 54 and pulse damper 56) is spread over multiple columns 10A, 10B, 10N. That is, a single parallel chromatography system 50 having common solvent delivery components and several (i.e., “N”) separation columns is substantially less expensive than a comparable (“N”) number of chromatography systems each having discrete solvent delivery components.
One alternative to using multiple discrete columns 10A, 10B, 10N in the system 50 is to utilize a microfluidic separation device having multiple separation columns that permit multiple chromatographic separations to be conducted in parallel, such as the device 100 illustrated in FIGS. 4A–4B. Preferably, such a device 100 contains multiple (e.g., eight) columns 145A–145X and is substantially planar to permit easy interface with one or more flat gasketed surfaces (not shown) for mating with solvent delivery components, detectors, and other desirable operational elements. This permits the device 100 to be interchanged rapidly as compared to the use of tubular columns (e.g., columns 10A, 10B, 10N) having conventional threaded fittings. Yet another device 400 having twenty-four separation columns 439A–439X permitting even higher throughput is illustrated in FIG. 5 and FIGS. 6A–6E. Further details regarding these microfluidic separation devices 100, 400 are provided herein.
While using common solvent delivery components with multiple separation columns (e.g., either discrete conventional columns 10A, 10B, 10N or multiple columns 145A–145X, 439A–439X integrated within a unitary device 100, 400) provides certain cost advantages, it also presents new issues compared to the use of single-column systems such as the chromatography system 30 illustrated in FIG. 2. One issue is accounting for different rates of fluid flow through each column in a multi-column system, particularly in low-flow environments not well-suited for inferential flow measurement. Generally, precision pumps used with liquid chromatography systems are of the positive displacement variety, and—assuming negligible leakage of the pump seals—such pumps permit liquid flow to be calculated by multiplying the number of pump strokes in a given period by the volume displaced by each stroke. When a positive displacement pump is coupled to a single column (e.g., a column 10 in the system 30 illustrated in FIG. 2), the rate of fluid flow through the column 10 is simply equal to the output of the pump.
When a common pump supplies multiple columns (e.g., columns 10A, 10B, 10N, 145A–145X, or 439A–439X), however, it is difficult to predict the flow through each individual column—even if the columns appear substantially identical—since impedance to fluid flow varies somewhat from one column to another and fluid flow will be biased toward the column(s) with the least fluidic impedance. That is, even when columns are carefully selected in an attempt to match their impedances, variations between columns will inevitably cause backpressure differences that in turn cause flow rate differences between fluid streams. Such flow variations may be caused by imperfect operation of any flow splitter(s) disposed upstream of the multiple columns, as well as by slight differences in column packing density, column geometry, and interfaces with the columns (e.g., threaded fittings within a chromatography system employing tubular columns or gasketed surfaces within a system employing substantially planar multi-column microfluidic devices).
To further complicate matters, many commonly employed chromatographic techniques utilize a solvent profile that changes with time. For example, common reverse phase chromatographic techniques use gradient elution wherein two individually controllable pumps supply two different mobile phase fluids (e.g., water and an organic solvent) that are mixed upstream of the separation column. (Contrast isocratic elution in which the solvent concentration remains constant so only a single pump is needed). Typically, separations in reverse phase chromatography depend on the reversible adsorption/desorption of solute molecules with varying degrees of hydrophobicity to a hydrophobic stationary phase. Thus, in a parallel multi-column system employing a common set of pumps for performing gradient elution, the presence of a changing solvent concentration exacerbates the difficulty of ensuring that identical mobile phase conditions (i.e., including both flow rate and concentration) are provided to each column at the same time.
Yet another potential source of variability in common-fluid-supply parallel HPLC systems employing low-pressure injection may be caused by the application of a pressure ramp to the columns following sample injection. Conventional HPLC systems, which use a loop injection valve for supplying sample to a column, permit sample introduction without depressurizing the column. In certain other systems, such as the planar multi-column systems described below, however, samples may be injected onto columns at low pressure (e.g., atmospheric pressure), followed by a pressure ramp up to a desirable operating pressure. Pressure ramps are inherently transient conditions, and can lead to unpredictable flow patterns in a common-fluid-supply parallel separation system before steady-state flow conditions are attained. These unpredictable flow patterns exacerbate column-to-column variability
Due to the difficulty of providing identical mobile phase conditions to each column in a parallel separation system with a common fluid supply, seemingly identical columns tend to perform differently. That is, if an identical sample mixture is provided to multiple columns in a common-fluid-supply parallel separation system with common mobile phase supply pumps for providing gradient conditions, individual species exhibit different retention times from one column to another. These different retention times are not merely proportional to flow. Applicants have confirmed this proposition experimentally by adding an unretained component (species that does not interact with the stationary phase and can be detected downstream to infer flow rate through a column) to the sample and simply correcting the retention times of unretained peaks. The simple phase shift based on flow rate did not yield nearly identical retention times from one column to another. As a result of the different performance characteristics of seemingly identical columns in a parallel separation system with a common fluid supply, it is difficult to compare analytical results obtained from one column to another.
The output of a chromatographic separation is a plot called a chromatogram, which is a graphical or other representation of the response of a detector to a property of the effluent versus time. For example, if UV-visible (“UV-VIS”) detection is used, then a chromatogram may include a plot of absorbance units versus time. An example of such a chromatogram resulting from separation of mixture of uracil and four parabens (methyl, ethyl, propyl, and butyl) (hereinafter, “Applicant's standard test mixture”) is provided in FIG. 7. Each of the components interacts differently with the stationary phase material (uracil does not interact at all), and therefore each has a different retention time (i.e., time corresponding to each peak in the chromatogram). More specifically, the retention time for a particular species is the time required for the species (initially contained in a sample) to travel from the injection port through the column to the detector. Retention times are used to identify components (species) in a particular sample. Due to the variation in performance among different separation columns, however, the retention time of a particular species in a sample eluted in a first column may be different from the retention time of the same species in the same sample eluted in a second column seemingly identical to the first. This is evident, for example, in FIG. 8, which provides superimposed chromatograms obtained using the same sample in multiple separation columns having a common mobile phase supply operated in gradient mode; although three species were eluted in each column, their retention times vary significantly. As evident from FIG. 9, in which the individual chromatograms from FIG. 8 have been time-shifted to a common retention time for the first unretained peak, a mere phase shift is generally insufficient to eliminate column-to-column variability for the remaining (retained) species. Since retention times are used to identify species within a sample, variation in retention times from one column to another increases the uncertainty of positively identifying individual species. It would be desirable to reduce this uncertainty; i.e., to provide an increased ability to discriminate between species with similar retention times, no matter which column in a common-fluid-supply parallel separation system is used.
There exist further reasons why it would be desirable to permit comparison between the outputs of different process regions (e.g., columns) in a parallel fluid processing system with a common fluidic input split among the process regions. One reason such comparison would be desirable is to enable the identification of “bad” experimental data, particularly those caused by difficulties with a particular process region. Conventionally, determining whether a particular process region is not working properly requires a skilled technician to compare and analyze data obtained using the same sample or reagent from multiple different process regions.
While certain performance variations among different process regions may be is relatively normal (e.g., different retention times among different separation columns in a multi-column system), the presence of more substantial differences (e.g., in chromatography, factors including peak shape and peak amplitude) can lead to a determination that a particular separation column is not functioning properly. It would be desirable to provide a process region evaluation method that could be automated, to not only reduce the time and skill required to evaluate process regions, but also rapidly validate experimental data.
In view of the foregoing, there exists a need for systems and methods for enhancing the utility of parallel fluid processing systems.